Anomalous pulmonary venous return is a common form of congenital heart disease though little is known about how the pulmonary veins are normally patterned. Semaphorins are guidance molecules that mediate repulsive cues for axons and blood vessels. We have generated two distinct null alleles of a poorly studied class III semaphorin, sema3d, which results in anomalous pulmonary venous return. Our data suggests that sema3d normally functions as a boundary, constraining migration of nascent pulmonary venous endothelium. The absence of sema3d allows for ectopic migration and patterning of pulmonary venous endothelial cells, leading to anomalous connections. These findings provide an altogether new description of the developmental cause of anomalous pulmonary veins that demands revision of common textbook explanations. In this proposal, we will test the hypothesis that sema3d binds directly to pulmonary venous endothelial cells causing repulsion. We will identify the functional sema3d receptor and the intracellular signaling pathways that result in repulsion, and we will examine how sema3d functionally interacts with Notch and Vegf during angiogenesis.